P89 RNA-seq CAR transcript analysis

Overview

Getting the data

Data presented below was generated and/or compiled from several sources:

• sample_metrics_data: sample annotation (e.g., donor ID, timepoint, etc.) as well sequencing & alignment metrics from RNA-seq processing
  
• sample_rapmap_data: read coverage measured across the length of the CAR transcript, based on mapping with the RapMap tool
• sample_salmon_data: abundance estimates (e.g., TPM, count) produced by the Salmon tool for CAR and several relevant transcripts when mapping to a modified human reference transcriptome (hg38)
• salmon_imgt_data: predicted/identified TCR junction sequences and alleles in single-cell libraries, as produced by assembly with Trinity followed by matching with IMGT High V-QUEST

load("data/sample_metrics_data.RData")
load("data/sample_rapmap_data.RData")
load("data/sample_salmon_data.RData")
load("data/sample_tcr_data.RData")

gff_file <- "data/annotation/carPlus.gtf"
xcripts_gtf <- import.gff2(gff_file)

Results

Visualizing CAR coverage & abundance

Bulk libraries from project P89

Single-cell libraries from project P89

Single-cell libraries from project P85

Inspecting trends in CAR coverage among single-cell P89 libs

Separating by QC outcome

• median_cv_coverage < 1
• mapped_reads_w_dups > 0.7

Separating by presence of functional TCR junction

Separating by presence of TCR (paired junction)

Inspecting abundance of non-CAR transcripts

Attempting to define CAR detection rules

Separating by CAR expression

log2(TPM +1) > 0 for CAR transcript

## Source: local data frame [6 x 3]
## Groups: car_expr_tpm [?]
## 
##   car_expr_tpm nz_cov n_distinct(lib_id)
##          (chr)  (lgl)              (int)
## 1 car_expr_tpm  FALSE                 18
## 2 car_expr_tpm   TRUE                 55
## 3 car_expr_tpm     NA                  1
## 4   no_car_tpm  FALSE                444
## 5   no_car_tpm   TRUE                 37
## 6   no_car_tpm     NA                  4

Quantification-based rule

log2(TPM + 1) >= 2.5 in any of CAR or EGFRt transcripts OR log2(TPM + 1) > 2 in all CAR or EGFRt transcripts

## Source: local data frame [6 x 3]
## Groups: car_expr_quant [?]
## 
##   car_expr_quant nz_cov n_distinct(lib_id)
##            (chr)  (lgl)              (int)
## 1 car_expr_quant  FALSE                107
## 2 car_expr_quant   TRUE                 58
## 3 car_expr_quant     NA                  2
## 4   no_car_quant  FALSE                355
## 5   no_car_quant   TRUE                 34
## 6   no_car_quant     NA                  3

Coverage-based rule

= 10 positions with > 0 reads in ANY of CD19scFv, T2A, or EGFRt

## Source: local data frame [5 x 3]
## Groups: car_expr_cov [?]
## 
##   car_expr_cov nz_cov n_distinct(lib_id)
##          (chr)  (lgl)              (int)
## 1 car_expr_cov  FALSE                  2
## 2 car_expr_cov   TRUE                 53
## 3   no_car_cov  FALSE                460
## 4   no_car_cov   TRUE                 39
## 5           NA     NA                  5

Revisiting splits

Separating by presence of functional TCR junction

Separating by presence of TCR (paired junction)